Cardioprotective effects of early versus late initiated antiretroviral treatment in adolescents with perinatal HIV-1 infection

Whether, and how, cardioprotective effects of antiretroviral treatment (ART) in adolescents with perinatal HIV infection (APHIV) vary with age at treatment initiation is unknown. We used magnetic resonance imaging to compare cardiac status between APHIV initiated on ART at < 5 years of age (early ART, n = 37) and ≥ 5 years of age (delayed ART, n = 34) versus HIV-uninfected peers (n = 21), reporting z-score mean differences adjusted for confounders. Relative to HIV-uninfected adolescents, APHIV with early ART had higher left ventricular (LV) global circumferential strain (GCS) [adjusted mean (95%CI) z-score: 0.53 (0.13, 0.92)] and maximum indexed left atrium volume (LAVi) [adjusted z-score: 0.55 (0.08, 1.02)]. In contrast, APHIV with delayed ART had greater indexed LV end-diastolic volume (LVEDVi) [adjusted z-score: 0.47 (0.09, 0.86)] and extracellular volume fraction [adjusted z-score: 0.79 (0.20, 1.37)], but lower GCS [adjusted z-score: −0.51 (−0.91, −0.10)] than HIV-uninfected peers. APHIV had distinct albeit subclinical cardiac phenotypes depending on ART initiation age. Changes in early ART suggested comparatively worse diastology with preserved systolic function while delayed ART was associated with comparatively increased diffuse fibrosis and LV dilatation with reduced systolic function. The long-term clinical significance of these changes remains to be determined.

risk factors 9 .Compounding this is the reality that perinatal HIV transmission in SSA is not expected to be eliminated before 2030 10 meaning that even modestly increased relative risks of cardiovascular disease will multiply into a large absolute case burden as more APHIV survive into adulthood.
However, our knowledge of the phenotypes and epidemiology of these perinatal HIV infection (PHIV) related cardiac forms remains incomplete, especially regarding the timing of ART.This, in turn, curtails our capacity to intervene preventively, let alone selectively.One major drawback to cardiovascular research in APHIV in SSA has been its almost exclusive reliance on echocardiography [4][5][6] notwithstanding the modality's limitations 11 .Cardiovascular magnetic resonance (CMR), with its multi-parametric capabilities, is the gold standard for cardiac volumetric and functional assessment 11 .Because of its capabilities to characterize myocardial tissue, CMR has been likened to a "virtual biopsy" 12 and is thus well suited to the challenge at hand.Unfortunately, CMR is inaccessible in the typical adolescent HIV care setting in SSA 13 .In response to these gaps, we conducted a CMR study to comprehensively characterize residual cardiac abnormalities in ART-experienced APHIV in Cape Town, South Africa.Our specific objective was to identify and quantify domain specific, i.e., geometry, systolic and diastolic function, mechanical deformation, and tissue composition, cardio-protective effects of early versus delayed ART in a high PHIV burden African setting.

Table 1.
Participants' baseline characteristics according to perinatal HIV infection and antiretroviral treatment status.Values are reported as mean (SD) or number (%). a Stunted = height-for-age z score < −2; underweight = weight-for-age z score < −2; wasted = BMI-for-age z score < −2; overweight/obese = BMI-for-age z score > 1.   ), all study measures of LV systolic function (ejection fraction, systolic strain and time to peak systolic strain) were collectively suggestive of comparatively lower function (albeit within normal range) in APHIV with delayed ART relative to their HIV uninfected peers.However, LV diastolic function was preserved as determined by measures like longitudinal [adjusted z score: −0.04 (−0.45, 0.73); p = 0.98] and radial [adjusted z score: 0.20 (−0.34, 0.57); p = 0.61] diastolic strain rate (Fig. 2).The distribution of absolute means (Table 3) was largely consistent with the adjusted z score based results.Of note, all the observed PHIV/ART related LV differences were of small-to-moderate size based on the z scores.

Discussion
In this CMR study, we identified distinct albeit subclinical cardiac phenotypes associated with early (< 5 years of age) and delayed (≥ 5 years of age) ART initiation among APHIV.Changes seen with early ART included relatively low LV mass, improved LV systolic function, and LA enlargement notwithstanding preserved LV diastolic mechanical deformation.On the other hand, delayed ART was associated with comparative LV chamber enlargement, reduced systolic function and greater interstitial fibrosis.These differences may be rooted in differing degrees of systemic inflammatory activation and immunosuppression correlated with ART timing in PHIV and, in turn, the extent and type of cardiac damage sustained in early life.Whether and how these difference might impact clinical cardiovascular trajectories of APHIV initiating ART at different ages remains to be determined.Our results, while consistent with previous reports of cardioprotection with ART in APHIV, also highlight the potential heterogeneity of these effects.This may have far-reaching clinical implications in the setting of SSA`s pediatric HIV epidemic.Currently available studies 3,5,6,14 of ART and the heart in Africa`s young persons with PHIV have almost exclusively included participants with late HIV diagnoses, delayed ART initiation, and/ or short duration of ART exposure.These studies found not infrequent cardiac functional and morphological abnormalities including LV hypertrophy (67% prevalence 15 ), dilatation (8% prevalence 1 ), systolic (5.5-33% prevalence 6,15 ) and diastolic (36% prevalence 16 ) dysfunction, and RV dilatation (29% 15 ), and often accompanied by symptoms of exertional dyspnea, leg swelling and cough, among others.In contrast, Mahtab et al. (2020) 5 reported very low prevalence of echocardiographic abnormalities (6.7% LV hypertrophy, 7.6% diastolic dysfunction, 0.2% systolic dysfunction) in a South African APHIV cohort.This is possibly the only African report to Our study did not enumerate cardiac abnormalities, and thus we cannot compare their prevalence with prior reports.However, there are notable differences in cardiac phenotype observed with either early or delayed ART in our study compared to previous reports.For example, delayed ART in our cohort was phenotypically marked by relative LV chamber enlargement, reduced systolic function and greater interstitial fibrosis.LV diastolic function was preserved as were LA and RV function and morphology.In other studies, LV enlargement and reduced LV systolic function with delayed ART were reported along with other abnormalities including LV hypertrophy, impaired LV diastolic function, RV dilatation and increased pulmonary artery systolic pressure 1,4,6,14,15 .Further, the cardiac changes seen with delayed ART initiation in our study were not associated with any symptoms and were, at best, of moderate size.This contrasts with other reports, mostly from SSA, where cardiac abnormalities in delayed ART frequently occurred with symptoms of heart failure.It is noteworthy that ART duration (median 8.1 years) with delayed ART in our cohort was much longer than in comparable studies.www.nature.com/scientificreports/Similarly, studies from HICs invariably found no clinically significant cardiac differences between those with PHIV and early ART initiation and their HIV uninfected counterparts 3,17,18 .The subclinical differences that have been described, at times contradictory, entail LV systolic strain, LV fractional shortening, and LV mass.Our observation of reduced LV mass with early ART echoes the results of a US study of children and adolescents early exposed to ART 19 .The comparatively greater peak systolic strain and shorter times to peak strain seen in APHIV with early ART are suggestive of hyperdynamic LV function.This has been previously noted in a similarly aged (mean age 15.3 years) cohort of APHIV undergoing echocardiographic examination in Kenya 20 .However, the notable difference is that the latter had late ART initiation (mean age 8.1 years) and high rates (53%) of uncontrolled HIV infection.Previously unreported, however, was our finding of relative LA enlargement.LA enlargement is often a marker of chronically elevated LA pressure, and signifies increased LV stiffness and diastolic dysfunction.However, formal LV diastolic functional assessment requires measurement of flow and velocities at the mitral annulus, mitral inflow and pulmonary veins 21 -which we did not evaluate-together with LA dimensions.
Nonetheless, the cardiac phenotype we observed in APHIV with early ART, i.e., preserved systolic function with likely impaired diastology, may mirror the shifts in epidemiology of HIV cardiomyopathy that have been observed in adult HIV care in HICs 22,23 .LV diastolic dysfunction, often with preserved systolic function, is now described in one in two (43-50%) 22,23 asymptomatic HIV infected adults on long-term suppressive ART.This in turn warrants concern about future heart failure (HF) risks, especially HF with preserved ejection (HFpEF).HFpEF is often preceded by LV diastolic dysfunction with normal systolic function and is the predominant manifestation of HF in adults with HIV.It is noteworthy that uptake of early infant diagnosis of HIV and linkage to pediatric ART are increasing across SS 24 .Thus, the prevalence and incidence of the cardiac phenotype associated with APHIV with early ART are likely to increase.Follow-up studies mapping the evolution and long-term outcomes of this cardiac phenotype are, therefore, urgently required.

Strengths and limitations
We believe that our study is among the first to deploy CMR in adolescents with PHIV in SSA.Previous studies have invariably used echocardiography, which despite its lower cost, ready availability, and ease of use, has well documented limitations in accuracy and reproducibility.CMR, in contrast, has superior spatial resolution, excellent inter-and intra-study reproducibility, no acoustic window dependency, and makes no geometric assumptions.A drawback, however, was lack of repeat imaging precluding us from knowing whether cardiac status was static, improving, or deteriorating.Similarly, the study`s cross-sectional design prevents causal inferences.Our small sample size was another limitation as it prevented examination of important subgroups like sex, and the estimation of precise results.Lastly, our HIV uninfected participants may not have been the ideal reference group given their comparatively high rates of overweight/obesity, and the potential for confounding.However, this limitation will continue to plague adolescent cardiac imaging research in Africa until normative CMR ranges are published.

Conclusions
We found residual albeit subclinical cardiac changes in South African APHIV on successful antiretroviral therapy suggesting that ART is cardioprotective.However, these cardioprotective effects are heterogenous and may depend on age ART initiation.Their long-term clinical significance remains to be determined, especially considering growing numbers of APHIV who are surviving into adulthood in Africa.

Methods
We followed the guidelines of the Strengthening the Reporting of Observational Studies in Epidemiology (STROBE) in the conduct and reporting of our analyses 25 .

Study population and setting
This was an observational cross-sectional study of participants enrolled in the ongoing prospective Cape Town Adolescent Antiretroviral Cohort (CTAAC).The CTAAC, described fully elsewhere 5 , enrolled young persons, then aged 9-14 years old (2012-2013), with PHIV and established on ART and a comparison group of similar age and sex HIV uninfected peers.Cohort members with PHIV and their HIV uninfected controls are all drawn from similar communities in Cape Town.CTAAC`s overall aim is to track the development of chronic diseases in children and adolescents with PHIV.For the current cross-sectional analysis, we approached and enrolled cohort members consecutively presenting for a scheduled study visit (Visit #9, 2018-2019) to complete a comprehensive CMR examination.Participants were eligible to participate if they were aged ≥ 13 years, had no history of clinical heart disease, no active systemic infections, no current use of anti-inflammatory therapies, and no known contraindications to CMR [e.g., gadolinium sensitivity or reduced glomerular filtration rate (≤ 30 ml/min/1.73m 2 )].

Image acquisition
As previously described, protocol-directed CMR was performed at 3 Tesla (Siemens, Magneton Skyra, Erlangen, Germany) 27 .The protocol included balanced steady-state free precession (bSSFP) for volumetrics and function, T1 mapping by Modified Look-Locker Inversion Recovery (MoLLI) sequence, T2 mapping using fast-low-angleshot (FLASH) sequence, and late gadolinium enhancement (LGE) using a phase sensitive inversion recovery (PSIR) sequence 10-15 min after gadolinium contrast administration.Imaging parameters for the sequences used were maintained between participants and are summarized in Supplementary Table S1.CMR image analysis was done offline, blind to PHIV/ART status, using proprietary software (CVI42, Circle Cardiovascular Imaging, Calgary, Canada) 28 .

Image analysis
LV end-diastolic volume (LVEDV), LV end-systolic volume (LVESV), LV ejection fraction (LVEF) and LV mass (LVM) were automatically derived from SAX views.Relative wall thickness (RWT) was calculated as twice inferolateral wall thickness divided by LV end-diastolic diameter, whereby both inferolateral wall thickness and LV end-diastolic dimension were measured in end-diastole from a SAX frame immediately basal to the tip of papillary muscle tips 28 .We assessed the tricuspid annular plane systolic excursion (TAPSE) in the LAX fourchamber view by measuring the distance travelled by the tricuspid annulus from end-diastole to end-systole.Basal, mid-ventricle, and apical SAX slices were manually contoured to outline the endocardium and epicardium, and generate maps for native T1, T2 and extracellular volume (ECV) estimation.We calculated the T2 signal intensity (SI) ratio as previously described 28 , and visually assessed the presence or absence of LGE.LV circumferential, radial, and longitudinal deformation parameters (global systolic strain, time to peak systolic strain, peak diastolic strain rate, and diastolic velocity) were analyzed by semi-automated feature tracking during diastole and systole from long axis (LAX) and SAX cine images.

Statistical analyses
A formal sample size calculation was not performed due to the exploratory nature of the study.Missingness in the key study variables was less than 2%, and thus we proceeded with complete case analysis.Where relevant, cardiovascular parameters were indexed to Mosteller body surface area (BSA) 29 to account for somatic growth, and are indicated by the postscript (i).Ninety-five percent confidence intervals (95%CI) were calculated based on bootstrap replications.Summary statistics are presented as mean (standard deviation, SD) or median (interquartile range, IQR) for continuous variables and number (percent) for categorical variables.Our primary exposure groups were HIV uninfected, APHIV with early ART, and APHIV with delayed ART.Comparisons by exposure were made using linear and quintile regression modeling, as appropriate.Differences in the distribution of blood pressure and standardized anthropometric measures were visually displayed.
There are currently no published reference standards for CMR-derived parameters for children and adolescents (< 20 years old) in SSA.We, therefore, derived z scores of cardiac variables for APHIV by referencing their HIV uninfected peers as normative.For a given variable, its z score was calculated by: (mean aphiv − mean hiv uninfected )/(SD hiv uninfected ).A z-score measures in SD units the distance of a raw score from the mean.Thus, a z score of 0.5 (or -0.5), for example, means that APHIV have a cardiac parameter that is 0.5 SDs above (or below) the HIV uninfected (i.e., control) group's mean.To quantify group differences in cardiac parameters, we classified the z scores as small (< 0.5), medium (≥ 0.5 and < 0.8), or large (≥ 0.8) using Cohen's criteria 30 .We fitted linear regression models to evaluate the association between PHIV/ART status and cardiac parameters (measured as z scores) after adjustment for age, sex, MAP and BMI.All statistical analyses were performed using Stata version 17.0 (StataCorp, College Station, TX) and R, version 3.6.3(R Foundation for Statistical Computing, Vienna, Austria).

Figure 1 .
Figure 1.Distribution of (A-C) age-standardized anthropometric measures and (D-F) blood pressure according to perinatal HIV infection and antiretroviral treatment status.P-values are estimated from unadjusted median quintile regression models referenced to HIV uninfected group.BFA BMI-for-age, HFA height-forage, WFA weight-for-age, MAP mean arterial blood pressure, SBP systolic blood pressure, DBP diastolic blood pressure.

Figure 2 .
Figure 2. z scores of left ventricular parameters according to antiretroviral treatment status among participants with perinatal HIV infection.Asterisk: adjusted for age, sex, mean arterial pressure (MAP) and body mass index (BMI).Abbreviations as elsewhere defined.Postscript (i) indexed to body surface area.

Figure 3 .
Figure 3. z scores of left and right ventricular and left atrial parameters according antiretroviral treatment status among participants with perinatal HIV infection.Asterisk: adjusted for age, sex, mean arterial pressure (MAP) and body mass index (BMI).Abbreviations as elsewhere defined.Postscript (i) indexed to body surface area.

Table 3
summarizes absolute mean values of left ventricular (LV) indices while Figs.

Table 2 .
HIV disease markers stratified by antiretroviral treatment status among participants with perinatal HIV infection.Values are reported as median (25th, 75th percentile) or number (%).PI protease inhibitors, NNRTI nonnucleoside reverse transcriptase inhibitors, NRTI nucleoside reverse transcriptase inhibitors, INSTI integrase strand transfer inhibitor.a Only one participant exposed to an integrase strand transfer inhibitor for total duration of 1.7 years.b Participant receiving 1 NRTI + 1 PI + 1 INSTI.c Detection limit ≥ 40 viral copies/mL.

Table 3 .
Participants' absolute mean values of cardiac parameters stratified by perinatal HIV infection and antiretroviral treatment status.LV left ventricle, LVM LV mass, LVEDV LV end diastolic volume, LVESV LV end systolic volume, LVEF LV ejection fraction, RWT relative wall thickness, GCS peak global circumferential strain, GLS peak global longitudinal strain, GRS peak global radial strain, DSR peak diastolic strain rate, SIR signal intensity ratio, ECV extracellular volume, LGE late gadolinium enhancement, LA left atrium, LAV LA volume, LAEF LA ejection fraction, RV right ventricle, RVEDV RV end diastolic volume, RVESV RV end systolic volume, RVEF RV ejection fraction, Postscript (i) indexed to body surface area.a P value for differences in mean (or proportion c ) between HIV uninfected versus PHIV with early ART initiation.b P value for differences in mean (or proportion c ) between HIV uninfected versus PHIV with delayed ART initiation.